GCMS (Gas Chromatograph Mass Spectrometer) In Drug Development: Principles, Applications, Case Studies and FAQs

GCMS In Drug Development

GCMS in Drug Development; It is a highly sensitive, accurate and reliable analytical technique, widely employed in drug development for both qualitative and quantitative analysis. Its exceptional sensitivity makes it particularly valuable for detecting and quantifying impurities, especially at trace levels, including nitrosamines and other genotoxic compounds.

In this article, I will provide skill-based insights into the principles, applications, advantages, limitations, case studies, and frequently asked questions related to GC-MS. By the end of this article, you will be well-equipped to develop GC-MS methods and predict the structure of unknown compounds during various stages of drug development.

GCMS

GC-MS is a unique combination of two powerful techniques. The first technique is the GC instrument with a capillary column, and the second technique is the mass detector. The function of GC is to separate different components from the sample mixture and send them one by one to the MS detector. The function of the MS detector is to break down each component into its different fragments (minor and major). These different fragments give structural information about the component. The major fragment is also used for quantification of the component.

Gas Chromatograph Mass Spectrometer

GCMS is the combination of a Gas chromatograph and Spectrophotometer

GCMS = GC + MS

Related: How To Control Impurities In Pharmaceuticals: Get Mastery In …

Principle of GC-MS

GC-MS is the integration of GC with a capillary column and an MS detector

1. GC with capillary column: The function of GC is to separate different components from the sample mixture and send them one by one into the MS
2. MS: The function of the MS is to break down each component into its different fragments (minor and major). These different fragments give structural information about the component. The major fragment is also used for the quantification of the component. The following are the main components of MS:
   • Interface: The function of the MS interface is to receive analyte from the capillary column and send it to the ion source.
   • Ion-source: The function of the ion source is to ionise the neutral molecules into their different fragments and send them to the Quadrupole. The pattern of this fragment is highly specific and acts as a fingerprint, which is used to structure characterisation or unknown compound identification.
   • Quadrupole: The quadrupole acts as a mass filter and separates ions based on the M/Z ratio. It is also called a mass analyser. It ensures that only a fragment of a specific mass travels towards the detector at a given time
   • MS Detector: The function of the Detector is to convert each fragment into a signal and send it to the data processor.
   • Data processor: The function of the Data processor is to convert all signals of an analyte into a mass spectrum

Types of GCMS ion source

The Electron ionisation (EI) and chemical ionisation (CI) are used in most of the GC-MS instruments:

Electron ionisation (EI)

It is also called hard ionisation, and it is widely used for structure elucidation. The molecule which enters into the EI-ion source gets electron bombarded of 70 EV. Due to this bombardment molecule breaks down into different fragments. The pattern of this fragment is highly specific and acts as a fingerprint, which is used for the structure characterisation of the unknown compound. A stable (M⁺) or an unstable (M*) molecular ion may form due to this bombardment

M +e^– → M⁺

M + e^– → M* →M¹⁺ or M²⁺ or M³⁺

Chemical ionisation (CI)

It is also called soft ionisation, and it is used for mass determination. In this process, methane gas is passed at high pressure in the CI source. Since methane has a very low proton affinity* and hence it can transfer a proton to any molecule, and that is the reason it is used in the CI source for ionisation of the molecule. The CI process may go by the following mechanism:

• CH₄  +  e-  →  CH₄⁺, CH₃⁺, CH₂⁺
• CH₄⁺  + CH₄   → CH₅⁺  + CH₃
• CH₃⁺  + CH₄   → C₂H₅⁺  + H₂
• CH₂⁺  + CH₄   → C₂H₄⁺  + H₂  and so on

Proton transfer

M +CH₅⁺  →[M-H]⁺ + CH₄ and so on

Note: Proton affinity for methane is 549/kJ/mol

What is the difference between EI and CI mode?

GCMS Spectra Representation

In GC-MS, the x-axis represents the m/z ratio, and the y-axis represents the relative intensity of the fragment.

SIM mode and MRM mode

SIM mode

SIM mode or selected ion monitoring mode allows the mass spectrometer to detect specific compounds with high intensity. In this mode single fragment is monitored. It is mainly used for quantification purposes.

MRM mode

MRM, or multiple reaction monitoring mode is used to collect data on different fragments of the molecule. It is used for structural characterisation purposes

Applications of GCMS

The following are the various applications of GCMS in the Pharmaceutical industry:

• Identification of unknown components: It is very helpful in the identification and characterisation of unknown impurities during drug development
• Identification of unknown organic volatile impurities: MS with GC-HS or GC-HS-MS Is very helpful in the identification and characterisation of organic volatile impurities or OVI
• Mass determination of known or unknown or known molecules: It is required for structural characterisation e,g. during characterisation of the standard
• Structure characterisation: MS in EI mode provides the specific pattern of fragments, which is very helpful for structural identification.
• Quantification of Genotoxic impurities at TTC level or very low level (at ppm or ppb level) in pharmaceuticals: It is widely used in the industries for the quantification of various nitrosamines at very low level impurities
• Content test: Quantification of residual pesticides
• Drug metabolism studies: GCMS is widely used for drug metabolism studies. By analysing biological samples such as blood and urine, scientists know about the drug metabolites, which is very helpful in understanding metabolic pathways
• Assay: Assay can be done by GC-MS. But it is not used for assay, considering the cost of analysis.

Apart from pharmaceutical industries, GCMS is the the following industries for various tests:

• Sports anti-doping: This is the main tool used in the anti-doping laboratory to test athletes’ urine samples for prohibited performance-enhancing drugs, for example, antibiotics and steroids
• Research and development centre
• Cosmetic industries
• Food industries
• Fragrance industries
• Wine and beverage industries
• Polymer industries
• Pesticides industries
• Environmental monitoring
• Biological analysis
• Forensic analysis

Advantages and Disadvanatges of GCMS

Advantages

The following are the advantages of the GC-MS:

• High sensitivity Instrument
• Accurate, precise and reliable result
• Structure characterisation of unknown compounds: The Structure of unknown compounds can easily be predicted using different fragments in EI mode or using the GSMS spectrum

Disadvantages

The following are the disadvantages of the GC-MS:

• High cost: It is a costly instrument, and hence, small industries can not afford it
• Not suitable for non-volatile material compounds: It is only suitable for volatile compounds, and non-volatile compounds can not be analysed
• Long analysis time: The System needs a longer time for stabilisation. Secondly, it takes more time to to stabilise the system after changeover from EI mode to CI mode and vice versa.
• Special skill: It requires the dedicated and well-trained person to operate the instrument. The person must have knowledge of organic chemistry.
• Needs extra pure chemicals and gases: It needs extra pure (G-MS grade) solvents and gases to perform the analysis. It increases the cost of analysis

Structure Characterisation By GCMS

Use the following steps to identify the structure:

• Write down all EI (electron ionisation) fragments of the unknown molecule
• Take the probable EI spectrum of the unknown compound from the EI library
• Write down all solvents or chemicals used at that stage in the process
• Now correlate the fragments, probable structure obtained from EI library and solvents or chemicals used in the process
• Conclude the result and make the report

Case Study:

An unknown compound that gives major fragments of 29D, 43D, 45D, 51D and 60D in EI mode. In CI mode, it gives the mass of 60D.

Interpretation

The EI spectrum predicts the closest structure of Acetic acid.

• Secondly, Acetic acid is also used in the process
• Therefore, the above masses are due to the following fragments of acetic acid:

Molecular ion (M⁺)

The molecular ion for acetic acid is typically observed at m/z 60: CH3COOH⁺ (m/z 60)

Loss of the hydroxyl group (COOH → CO)

One common fragmentation pathway is the loss of the -OH group from the carboxyl group. This produces an ion at: CH3CO⁺(m/z 43)

This fragment is stable and often one of the most prominent peaks in the EI mass spectrum. I

Loss of CO₂ (Decarboxylation)

Acetic acid can also undergo decarboxylation, losing a molecule of carbon dioxide (CO₂). This process is common for carboxylic acids. The resulting fragment ion is: CH₃⁺(m/z 15)

This is a methyl ion, a very stable species. Decarboxylation is a strong fragmentation pathway for carboxylic acids, and this peak is often observed.

Cleavage of the C–C bond (Formation of C₂H₅⁺)

Another fragmentation pathway involves the cleavage of the C–C bond in acetic acid. This produces an ethyl ion C_2​H₅⁺​(m/z 29)

Conclusion

The control, quantification, characterization, and identification of highly carcinogenic compounds would be extremely challenging – if not impossible – without the use of GC-MS. This is why GC-MS plays a crucial role in pharmaceutical research and development. Hopefully, this post has helped clarify the importance of GC-MS and deepened your understanding of its applications

You may also want to check out other articles on my blog, such as:

• What is the Analytical Method Validation?
• The 7 Key Differences Between Validation and Verification
• How to perform cleaning validation?
• What Is Forced Degradation Studies Of Pharmaceuticals and How To Perform?
• What Is Photostability Testing Of New Drug Substances: Learn in 5 minutes
• What is CSV (Computer System Validation): Learn in 12 Easy Steps

FAQs

Abbreviations

• GC: Gas chromatography
• HS: Head space
• MS: mass spectrometry
• EV: Electron volt
• EI: Electron ionisation
• CI: Chemical ionisation
• M/Z: Mass/charge

References

• Interpretation of mass spectra; Fred W. McLafferty
• Wikipedia